Method of inhibiting growth of tumor cells

ABSTRACT

This invention relates to a novel process for inhibiting the growth of malignant tumor cells in mammals, particularly the growth of multiple myeloma in humans, employing the alkaloid acronycine as the inhibiting agent.

CROSS-REFERENCE

This application is a continuation of my copending application Ser. No.720,711 filed Aug. 7, 1976, now abandoned, which was a division ofapplication Ser. No. 374,588, filed June 28, 1973, now U.S. Pat. No.3,985,899, which was a continuation of application Ser. No. 268,406,filed July 3, 1972, now abandoned, which was a continuation-in-part ofmy copending application Ser. No. 46,811, filed June 16, 1970, nowabandoned, which was a continuation-in-part of my then copendingapplication Ser. No. 840,114, filed June 18, 1969, now abandoned, whichwas a continuation-in-part of my then copending application, Ser. No.536,905, filed Mar. 23, 1966, now abandoned.

BACKGROUND OF THE INVENTION

Most of the agents useful in inhibiting the growth of malignant tumorcells discovered to date have either been alkylating agents, of thenature of the nitrogen mustards, or anti-metabolites for vitamins andnucleic acids, such as 8-azaguanine, 5-fluoronicotinic acid, and thelike. Recently a group of novel alkaloids, obtainable in pure form fromthe plant Vinca Rosea, have been found to be particularly valuable inthe treatment of leukemias and have even shown some activity againstvarious solid tumors. These alkaloids have a chemical structure which istotally unrelated to any known anti-tumor agent, and they apparently actin a manner distinctly different from such other agents.

It is an object of this invention to provide a method of inhibiting thegrowth of malignant tumor cells in mammals, specifically mice, whichmethod employs a previously known alkaloid not related structurally tothe Vinca alkaloids and having a quite different anti-tumor spectrumwhen compared with that of any of the anti-tumor agents heretoforeknown.

SUMMARY OF THE INVENTION

In fulfillment of the above and other objects, this invention provides amethod of inhibiting the growth of malignant tumor cells which comprisesadministering to a mouse, in whose body the malignant tumor is presentand proliferating, an effective quantity of the alkaloid acronycinerepresented by the following formula: ##STR1##

Acronycine, among other alkaloids, was obtained originally in smallquantity, from the bark of the Australian scrub ash or scrub yellowwood, Acronychia Baueri Schott, (also known as Bauerella AustralianaBorzi) by Lahey and co-workers [Australian J. Sci. Res., 2A 423-b(1949)]. A description of the physical properties of the purifiedmaterial is given in ibid, 3A, 593-614 (1950). The following preparationillustrates my method of obtaining acronycine from Achronychia BaueriSchott bark:

Preparation of Acronycine

About 1.5 kg. of coarsely ground bark of the Australian scrub ash,Acronychia Baueri Schott, were stirred with two 3.5-liter portions ofn-hexane in order to extract fatty material present therein. Thedefatted bark was then extracted with three 4-liter portions of diethylether. The ether extracts were combined, and the combined extracts wereconcentrated in vacuo to a volume of about 1 liter. Upon cooling, about11.5 g of yellow-gold crystals precipitated from the concentrate. Thesecrystals were separated by filtration, and were proven to be a mixtureof the known alkaloids acronycine and melicopine by means of x-ray,ultraviolet, and infrared spectra and by thin layer chromatography.Recrystallization of the crystal mixture from methanol, employed at therate of 10 ml. of methanol per gram of crystals, yielded four successivecrops of crystals as follows: 1.29 g. of melicopine; 2.60 g. of slightlyimpure acronycine; 1.39 g. of a mixture of acronycine and melicopine;and 1.69 g. of slightly impure acronycine. The second and fourth crystalfractions, which were substantially pure acronycine, were combined andrecrystallized from methanol at the rate of about 8 ml. per gram ofcrystalline material. About 4.14 g. of acronycine were obtained thereby.Acronycine thus purified was one-spot material upon thin-layerchromatography; i.e., contained no other alkaloids.

Properties of Acronycine

Pure acronycine produced as above had the following physicalcharacteristics:

Crystals

The crystals obtained from methanol were opaque yellow coarse particleswhose precise crystalline form could not be determined. The crystalsobtained from ethanol were clear yellow needles occurring in fan-shapedclumps. The crystals from acetone were monoclinic rods.

Melting Point

Acronycine had the following melting point: 174°-176° C.

Elemental Analysis

Calcd. for C₂₀ H₁₉ NO₃ : C, 74.74; H, 5.96; N, 4.36; O, 14.94. Found: C,74.76; H, 6.19; N, 4.29; O, 15.01.

Ultraviolet Spectrum

The ultraviolet spectrum had peaks at the following wave lengths withthe extinction coefficient for each peak given immediately thereafter inparentheses:

λmax (EtOH)=280 mμ (log ε=4.60), 291 mμ (log ε=4.54), 304 mμ (logε=4.28), 392 mμ (log ε=3.84).

Infrared Spectrum

The infrared spectrum of acronycine is given in FIG. I.

X-Ray Crystallography

The following table gives x-ray crystallography data for acronycine. Thetable lists the wave length, d, at which absorption occurs, and theintensity, I/I₁, of the particular band.

                  TABLE I                                                         ______________________________________                                        d     I/I.sub.1                                                                             d       I/I.sub.1                                                                           d    I/I.sub.1                                                                           d     I/I.sub.1                        ______________________________________                                        9.85  1.00b   5.67    .15   4.10 .06   3.57  .02                              7.36  .06     5.48    .20   4.02 .06   3.41  .30                              6.88  .30     5.29    .15   3.88 .08   3.18  .30                              6.50  .06     4.80    .15   3.72 .04   3.01  .04                              5.87  .20     4.50     .06b 3.64 .75   2.91   .06b                            2.70  .02     2.25    .04   2.01 .02                                          2.64  .02     2.15    .02   1.99 .02                                          2.53  .02     2.14    .02   1.86 .02                                          2.33  .04     2.07    .04                                                     2.28  .02     2.05    .04                                                     ______________________________________                                    

Acronycine has shown excellent anti-tumor activity against the followingvarieties of transplanted tumors in mice: adenocarcinoma, Ad-775: plasmacell myeloma, X-5563; Mecca lymphosarcoma, MLS; B-82 leukemia; C-1498myelogenous leukemia; Sarcoma 180; Ridgeway osteogenic sarcoma; Shionogicarcinoma 115; and high-malignancy alone. Indefinite survivors, micewhich survive 45 days or longer after inoculation with the tumor cellsin the case of leukemias, or who survive indefinitely aftertransplantation of the tumor cells in the case of sarcomas orcarcinomas, have been found with each of the above tumors, aftertreatment for 10 days with acronycine. The C-1498 myelogenous leukemiaand Mecca lymphosarcoma are both metastasizing tumors in mice.Acronycine is the first alkaloid found to adversely affect the progressof the transplanted tumor C-1498 myelogenous leukemia. With the plasmacell myeloma, X-5563, there is an abnormal protein found in theτ-globulin fraction of the blood of mice bearing this tumor. Treatmentwith acronycine has produced not only an inhibition of the X-5563myeloma with a decrease in tumor size in all animals, plus 8 out of 10indefinite survivors in one test, but also has decreased the amount ofthe abnormal protein in the blood.

Another interesting activity of acronycine is its effectiveness againstthe Shionogi carcinoma 115. This carcinoma is an androgen-dependenttumor, and in mice there is found a 100 percent inhibition of the tumorwith indefinite survivors. Treatment in the case of the mice implantedwith Shionogi carcinoma 115 was begun 5 days after transplantation.

Acronycine has demonstrated its anti-tumor activity when administered tomice by the oral, intraperitioneal, intravenous and subcutaneous routes;the compound is administered orally either in a telescoping gelatincapsule or as a pill after admixing with customary pill-makingingredients. For intraperitioneal or subcutaneous administration, thecompound is ground with Emulphor (a polyoxyethylated fatty acid which iswater miscible and nontoxic when diluted 1:10 with either sterile wateror sterile physiological saline solution). The Emulphor suspension isadministered as such or can be diluted with water. For intravenousadministration, the Emulphor suspension is diluted 1:10 withphysiological saline.

If an oral dosage form is to be used, the nature of the excipient ismost important in that, with certain excipients such as talc, there is alowered absorption of the drug from the intestinal tract. I prefer touse an excipient such as starch which does not interfere with drugabsorption in the intestine. In addition, there is better absorption ofacronycine if the drug is micronized before mixing with starch. In fact,a preferred preparation of our dosage form is one in which a solution ofthe drug is sprayed onto the starch or slurried with the starch. In eachcase, the solvent is removed by evaporation. The acronycine is presentin an amorphous form on the starch.

The following preparations will serve to illustrate the novelpharmaceutical corporation in unit dosage form for oral administrationuseful in the tumor-suppressing process of this invention.

PREPARATION I

0.5 g. of micronized acronycine (90 percent of the acronycine had a sizeof 10 microns or less) was mixed thoroughly with 2.29 g. of starch. Theempty telescoping gelatin capsules were filled with 0.27 g. each of theabove mixture.

PREPARATION II

0.5 g. of acronycine were dissolved in 250 ml. of ethylenedichloride.The resulting solution was slurried with 2.5 g. of starch and thesolvent removed by evaporation. Empty telescoping gelatin capsules werefilled with 0.30 g. each of the above mixture.

PREPARATION III

Telescoping gelatin capsules were each filled with the followingmixture: 100 mg. acronycine, thoroughly milled; 166 mg. lactose; 166 mg.starch; and 8 mg. polyoxyethylene sorbitan monooleate.

Another useful dosage form for administration of acronycine by the oralroute comprises a co-precipitate of acronycine and polyvinylpyrrolidone,as described in the patent application of W. D. Walkling, filed June 16,1970. These acronycine-PVP co-precipitates can be prepared by dissolvingeach of the components in ethanol, mixing the solutions in such amountas to obtain the desired ratios of acronycine and PVP in theco-precipitate and then removing the solvent by evaporation in vacuo.Particularly useful co-precipitates are those containing 1 part ofacronycine to 5 or 10 parts of PVP (M.W.=40,000). These co-precipitateshave a markedly greater stability than acronycine itself, being up to 15to 25 times as soluble.

The customary mode of administration of acronycine to mice is to givethe drug at a predetermined dosage level on each of ten successive days.The dosage regimen can vary from 15-100 mg./kg. per day by theintraperitoneal and subcutaneous routes and from 30-80 mg./kg. per dayper mouse by the oral route. The toxicity of acronycine marks the upperlimit for the daily dose.

Although the standard treatment regimen involves administering thelife-prolonging drug to mice 24 hours after the time when the tumorcells are inoculated or transplanted into the mouse, acronycine has theunusual property of being effective in prolongingthe life of tumor-cellinoculated mice when treatment is begun as late as 3, 5 or 6 days afterthe day of inoculation. This finding is of particular interest withregard to the treatment of C-1498 leukemia, where the percentprolongation of life is still appreciable even though administration ofacronycine, by either the intraperitoneal or oral route, is begun aslate as 6 days after inoculation with the leukemic cells. Similarresults have also been found in treating X-5563 myeloma. It should benoted that ordinarily, untreated mice die from the tumor in a periodvarying from 14-18 days after inoculation; that is to say, a treatmentbeginning the sixth day after inoculation could be considered to havebegun after one-third to one-half of the life span remaining to theanimal has already passed.

Acronycine is administered orally or subcutaneously to humans in dosageamounts varying from 15-2000 mg. per treatment day or at the rate of50-100 mg./kg. per treatment day, for a period of from 10 to 20 days. Auseful dosage regimen for humans consists of 800 mg. per day in 8divided doses.

The use of acronycine in combating multiple myeloma is shown by thefollowing case histories: A male patient diagnosed as suffering frommultiple myeloma was treated with 200 mg. per meter squared of bodysurface of acronycine per day for 50 days. The patient showed notoxicity from the drug. There was a cessation of pain in the spine andthe electrophoretic M protein had decreased 1.5 g. percent.

A second patient was treated for 90 days for multiple myeloma at thesame dose rate as above. The patient showed subjective improvementmarked by an absence of bone pain. Bony defects noted on X-ray had notchanged during the period of treatment.

Little is known about the mechanism of action of acronycine ininhibiting the growth of tumor cells, but it has been ascertained thatthe compound does not operate via mitotic arrest.

I claim:
 1. A method of treating multiple myeloma in humans whichcomprises administering acronycine to a patient suffering therefrom inan oncolytic amount.
 2. A pharmaceutical preparation in dosage unit formadapted for oral administration to achieve an anti-tumor effect inmultiple myeloma comprising per dosage unit from 15-100 mgs. ofacronycine and from 0.165 to 0.3 g. of starch.
 3. A pharmaceuticalpreparation in accordance with claim 2 in which 90 percent of theacronycine has a particle size of 10 microns or less.
 4. Apharmaceutical preparation in accordance with claim 2 in which theacronycine is present in amorphous form.